Microfluidic devices with new inner surfaces

ABSTRACT

A microfluidic disc having one or more enclosed microchannel structures, and the microchannel structures are intended to be used for transport of transporting liquids. The device is characterized in that at least a part of the inner walls of each of one or more microchannel structures are treated with a gas plasma having one or more organic precursor compounds.

This Application claims priority to U.S. Provisional Application No.60/371,080 filed on Apr. 9, 2002, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

I. Field of Invention

The present invention concerns a microfluidic device that has innersurfaces with chemical surface characteristics that have been introducedusing gas plasmas having one or more organic precursor compounds.

II. Related Art

A number of different techniques for modifying substrate surfaces arewell known. One common method is to subject a substrate surface, forinstance made in plastics, to various forms of plasma treatment (Chan etal., Surface Science Reports 24 (1996) 1–54; and Garbassi et al.,Polymer Surfaces—From Physics to Technology, John Wiley (1998) 238–241).This is done in a plasma reactor, which is a vacuum vessel containing agas at low pressure (typically 10 to 1000 mTorr). When a high frequencyelectric excitation field is applied over the reactor, a plasma (alsocalled glow discharge) is formed, containing reactive species like ions,free radicals and vacuum-UV photons. These species may react with otherspecies and/or with the surface and cause a chemical modification of thesubstrate surface with properties depending on the nature of the gas andon the plasma parameters. Gases like oxygen and argon are typically usedfor hydrophilization and/or adhesion improvement on plastics, whilevapors of organic precursor compounds can be used to apply thin coatingsfor a number of different purposes (Yasuda, Plasma Polymerization,Academic Press 1985).

Previously, vapors of organic precursor compounds have been used toproduce surfaces that are wettable by aqueous liquids but thehydrophilicity has been moderate and not utilized to facilitatetransport of aqueous liquids, in microchannels. In some cases, theprimary goal has been to introduce coats that have a low non-specificadsorption, for instance of proteins and/or other biopolymers and/orother bioorganic molecules. See for instance discussions U.S. Pat. No.5,153,072 (Ratner et al.), U.S. Pat. No. 5,002,794 (Ratner et al.), U.S.Pat. No. 6,329,024 (Timmons et al.), U.S. Pat. No. 5,876,753 (Timmons etal.), EP 896035 (Timmons et al.). Strictly hydrophobic surfaces havealso been produced. See for instance U.S. Pat. No. 5,171,267 (Ratner etal.).

WO 0056808 (Ocklind, Larsson and Dérand, Gyros AB) describesmicrofluidic devices comprising hydrophilic microchannel structuresdefined between two essentially planar substrates that are apposed.Before being apposed the surface of at least one of the substrates hasbeen hydrophilized in gas plasma, which comprises a non-polymerizablegas. The surfaces obtained are hydrophilic and can be coated subsequentto gas plasma treatment in order to introduce further functionalities.

WO 9958245 (Larsson et al.) and WO 97 21090 (Mian et al.) are examplesof publications that in general terms suggest microfluidic devices inwhich the inner surfaces of the microchannel structures have been madehydrophilic by gas plasma treatment, coating of hydrophobic surfaceswith hydrophilic polymer, etc.

BRIEF SUMMARY OF THE INVENTION

A first object of the invention is to present a surface modificationmethod. Accordingly, the first aspect of the invention is a method forthe manufacture of a microfluidic device to introduce a predetermineddegree of wettability (hydrophilicity and/or hydrophobicity) on an innersurface of said microchannel structures. The method is characterized incomprising the steps of: (i) providing two essentially planar substrates(I and II); (ii) placing either one or both of the substrates in a gasplasma reactor, and creating within said plasma reactor a gas plasmacontaining an organic precursor compound, said organic precursorcompound and the conditions in the reactor being selected such that acoat of the predetermined degree of wettability is formed on a selectedpart of the surface of the substrate/substrates; (iii) removing thesubstrate/substrates from the plasma reactor; (iv) adhering the surfaceof substrate I to the surface of substrate II so that at least anenclosed section of each of microchannel structures are formed betweenthe two surfaces; and (v) optionally joining further planar substratesto complete the microchannel structures. In the simplest variantcomplete enclosed microchannel structures are defined between substrateI and II.

A second object of the invention is to provide new surface modificationsthat have a sufficient wettability combined with a sufficiently lownon-specific adsorption for a reliable and reproducible mass transportand processing of reagents by a liquid flow through a microchannelstructure. This object, thus, aims at optimizing wettability andanti-fouling in relation to each other.

A third object is directed to a microchannel structure that is presentin a microfluidic device and comprises two or more different functionalparts, at least one of which comprises inner surfaces of a sufficienthydrophilicity for a liquid aliquot to penetrate completely thefunctional part by capillary force once having wetted the entrance ofthe part. The demand for a sufficiently low non-specific adsorptionremains.

A fourth object is to accomplish a microfluidic device comprising coatsthat can be stored for ≧7 days, such as ≧30 days, while retaining theintended functionality of the surface, i.e., the surface may still beused for the intended purpose (=is essentially unchanged).

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings.

FIG. 1 shows Total Internal Reflection Fluorescence (TIRF) withFluorescence-5-isothiocyanate-bovine serum albumin (FITC-BSA) onuntreated Polycarbonate (PC) (squares), and on PC treated with diglyme(24 W) in the plasma reactor (circles). Protein solution (400 ppm)enters the flow cell (filled arrow) and is replaced by PBS buffer(dashed arrow).

FIG. 2 shows TIRF with FITC-BSA on PC treated with diglyme (24 W), andallylic alcohol (12 W) in the plasma reactor. Protein solution (400 ppm)enters the flow cell (filled arrow) and is replaced by PBS buffer(dashed arrow).

FIG. 3 shows TIRF with FITC-BSA on PC treated with ethylene glycol vinylether-plasma (24 W) in the Gyros reactor. Protein solution (400 ppm)enters the flow cell (filled arrow) and is replaced by PBS buffer(dashed arrow).

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used herein, the use of the word “a” or “an” when used in conjunctionwith the term “comprisng” in the sentences and/or the specification maymean “one,” but it is also consistent with the meaning of “one or more,”“at least one,” and “one or more than one.”

As used herein, the term a “microfluidic device” typically comprisesone, two or more microchannel structures, which are defined between twoessentially planar and parallel substrates that are apposed to eachother. Thus, either one or both of the two substrate surfaces thatdefine the microchannel structures comprise microstructures in the formof grooves and/or projections such that the microchannel structures canbe formed when the two surfaces are apposed. The device is microfluidicin the sense that one or more liquid aliquots can be transported betweendifferent functional parts of the individual microchannel structures inorder to process the aliquots. The liquid aliquots are in the μl-rangewith preference for the nl-range. The purpose of the transport is tocarry out predetermined process protocols, for instance for assaying oneor more constituents of a sample aliquot or to synthesize an organic oran inorganic compound. The liquid aliquots are typically aqueous, i.e.,based on water and mixtures between water and water-miscible organicsolvents.

As used herein, the term “microchannel structure” relates to thestructure that is defined between the surfaces of two or more planarsubstrates that are layered on top of each other. If different sectionsof a microchannel structure are defined between different pairs ofplanar substrates, there typically are holes in the substrates so thatthe sections are in communication with each other. Either one or both ofthe surfaces that are to define a section of a microchannel structurecomprises microstructures such that the desired section of amicrochannel structure will be formed when the surfaces are joinedtogether. Separate microchannel structures may be defined betweenadditional essentially planar substrates.

As used herein, the term “plurality” means two, three, four, five ormore microchannel structures. Preferably “plurality” means that thenumber of microchannel structures on the microfluidic device is ≧10,such as ≧25 or ≧90 or ≧180 or ≧270 or ≧360.

As used herein, the terms “microchannel”, “microconduit”, etc.,contemplate that a channel structure comprises one or more cavitiesand/or channels/conduits that have a cross-sectional dimension that is≦10³ μm, preferably ≦0.5×10³ μm or ≦10² μm. The lower limit for crosssectional dimensions is typically significantly larger than the size ofthe largest constituent of a liquid that is to pass through amicrochannel of the innovative device. The volumes ofmicrocavities/microchambers are typically in the nl-range, i.e., ≦5000nl, such as ≦1000 nl or ≦500 nl or ≦100 nl or ≦50 nl or ≦25 nl. Thisdoes not exclude larger chambers/cavities, for instance in the intervals1–1000 μl, such as 1–100 μl or 1–10 μl which typically are directlyconnected to inlet ports and intended for application of sample and/orwashing liquids.

As used herein, the term “microformat” means that one, two, three ormore liquid aliquots that are transported within the device are withinthe intervals specified for the microchambers/microcavities.

As used herein, the terms “non-specific adsorption” and “fouling”, whichare interchangeable, mean undesired adsorption of compounds to innerwalls of the microchannel structures. The terms may also includeinactivation of bioactive compounds by the walls, for instancedenaturation of proteins. The compounds are present in the liquid usedand are primarily reagents. For aqueous liquids the reagents may beproteins and/or other biopolymers and/or other bioorganic and syntheticorganic compounds.

As used herein the term “anti-fouling” refers to reduction innon-specific adsorption (undesired adsorption) of reagents compared to areference surface that in the context of the invention is the surfacebefore being treated in a gas plasma comprising an organic precursor.

As used herein, the term “analytes” are also included in the term“reagent”.

As used herein, the term “wettable” refers to a surface having a watercontact angle that is ≦90° (hydrophilic surface).

As used herein, the term “non-wettable” refers to a surface that has awater contact angle ≧90° (hydrophobic surface).

As used herein, the term “wettability” refers to the “degree ofwettability” and may include a highly wettable surface, a highlynon-wettable surface or any variation therebetween.

As used herein, the term “predetermined degree of wettability” refers tothe wettability of a coat that is important for the function of amicrochannel structure. The predetermined degree of wettability mayimply a wettable surface that will allow for capillary flow, anon-wettable surface that will act as a valve, a vent, an anti-wickingmeans, etc. Typically, the expression means that the wettability of thecoat is different from the wettability of the surface without the coat.

As used herein, the term “organic precursor” refers to an organiccompound that forms reactive species in a gas plasma.

As used herein, the term “masking” refers to placing a removableprotective coat/mask on surface parts that are not to be coated by thesubsequently applied coating method.

II. Method of Manufacture

During the last decade sophisticated microfluidic devices have appearedwith the goal to fully integrate complete process protocols inminiaturized form. This means integration of all steps of a protocolfrom sample preparation to recording of the results in one and the samemicrochannel structure. Thus, it is advantageous if the same kind ofequipment is used to produce surfaces corresponding to a spectra ofchemical surface characteristics, for instance from extremelyhydrophobic to extremely hydrophilic surfaces, and preferably withanti-fouling properties.

The present inventors have recognized that the above-mentioned objectscan be achieved by treating the channel surfaces with gas plasma, whichcomprises one or more organic precursor compounds in gas form. Theobtained surface characteristics (for instance hydrophilicity orhydrophobicity) is determined by the selection of the organic precursorcompound and/or the process parameters applied to create the gas plasmaas outlined below.

Accordingly the first aspect of the invention is a method for themanufacture of a microfluidic device of the kind described above inorder to introduce a predetermined degree of wettability (hydrophilicityand/or hydrophobicity) on an inner surface of said microchannelstructures. The method is characterized in comprising the steps of: (i)providing two essentially planar substrates (I and II); (ii) placingeither one or both of the substrates in a gas plasma reactor, andcreating within said plasma reactor a gas plasma containing an organicprecursor compound, said organic precursor compound and the conditionsin the reactor being selected such that a coat of the predetermineddegree of wettability is formed on a selected part of the surface of thesubstrate/substrates; (iii) removing the substrate/substrates from theplasma reactor; (iv) adhering the surface of substrate I to the surfaceof substrate II so that at least an enclosed section of each ofmicrochannel structures are formed between the two surfaces; (v)optionally joining further planar substrates to complete themicrochannel structures. In the simplest variant, complete enclosedmicrochannel structures are defined between substrate I and II.

Microchannels are typically defined by a limited number of well-definedwalls, for instance a bottom wall, a top wall and two sidewalls. Thesewalls may derive from different substrates. Locally at least the wallsderived from the same substrate are wettable/non-wettable to the sameextent. In the case the surface characteristics of a channel is intendedto facilitate liquid transport, and the walls derived from one of thesubstrates is non-wettable this can be compensated if the wall(s)derived from the other substrate is(are) sufficiently wettable (i.e.,has/have a sufficiently low water contact angle).

In order to facilitate good transport of a liquid between differentfunctional parts of the inventive microfluidic devices, the liquidcontact angle in the individual parts should primarily be wettable,preferably with a water contact angle ≦60° such as ≦50° or ≦40° or ≦30°or ≦20°. Local surface breaks that are to be used for valving and/oranti-wicking, for instance, are important exceptions from this generalrule. Local surface breaks are typically non-wettable with water contactangles ≧90°, such as ≧100° or ≧110° or ≧120°. Typically the differencein wettability (in water contact angles) between a local surface breakand a bordering surface are ≧50°, such as ≧60° or ≧70°. All figuresrefer to values obtained at the temperature of use, typically 25° C.,and with water as the liquid.

One important problem with respect to microfluidic devices is to obtainsurfaces with a sufficient hydrophilicity to support liquid transportthrough a microchannel structure combined with a sufficiently lownon-specific adsorption (anti-fouling) of reagents in order toaccomplish reliable and reproducible results. The severity of thefouling problem (nonspecific adsorption) increases with the surface tovolume ratio, i.e., it increases when a cross sectional dimensiondecreases, for instance from ≦1000 μm to ≦100 μm to ≦10 μm and/or from≦1000 μl to ≦100 μl to ≦10 μl to ≦1 μl to ≦100 nl to ≦50 nl. Even if itis often said that hydrophobic surfaces have prominent non-specificadsorption there are numerous systems for which also hydrophilicsurfaces have a disturbing non-specific adsorption.

A. Additional Steps and Variations

Between steps (i) and (ii), (ii) and (iii) and/or (iii) and (iv) theremay be one or more additional steps for introducing one or more surfacemodifications with characteristics that are different from the coatintroduced in step (ii). These additional steps may involve (a) a gasplasma treatment utilizing the same or another precursor compound and/orthe same or other conditions, and/or (b) some other coating procedure.Depending on the kind of surface modification, alternative (a) may becarried out without removing and re-inserting the substrate/substratesfrom/into the gas plasma reactor.

If only a part of a substrate surface is to be coated in step (ii) or inany of the additional steps, appropriate masking and/or unmasking may bedone before or after such a coating step (including sequence of steps).Parts that are masked/unmasked may be present in either one or both ofthe substrate surfaces, for instance on a part comprisingmicrostructures. Washing steps may be included between steps ifappropriate.

One variant of step (ii) is to introduce a coat that is wettable(hydrophilic) and/or has a pronounced resistance to non-specificadsorption (=anti-fouling) on a major part of the microstructured partof the surface. Microstructured areas that are not going to be coated inthis step are typically masked. The precursor compound and the plasmaconditions for the gas plasma are selected as outlined below. After step(ii) and unmasking, the uncoated areas thus exposed may be furtherprocessed, for instance to render them non-wettable (hydrophobic) inorder to create passive (non-closing) valves and/or anti-wicking meansand/or inlet or outlet vents to ambient atmosphere. These kinds offunctionalities are illustrated in WO 9958245 (Larsson et al., GyrosAB), WO 0185602 (Larsson et al., Gyros AB & Åmic AB), WO 0146465(Andersson et al., Gyros AB), and WO 02074438 (Andersson et al., GyrosAB), which are incorporated herein by reference. In the case an uncoatedarea as such provides a sufficiently low wettability (i.e., arenon-wettable), the surface at these positions may be used directly as avalve and/or as an anti-wicking means and/or as a vent after step (iv)without any extra processing. Many times, however, it is moreappropriate to make these non-treated areas more non-wettable (increasethe hydrophobicity), for instance by inserting steps according toalternatives (a) or (b) between steps (ii) and (iv). In the casealternative (a) is selected, the precursor and gas plasma conditions areselected to give a non-wettable surface as known in the field and alsodiscussed below. Spraying or printing may also be utilized asalternative (b). See for instance WO 0185602 (Larsson et al., Gyros AB &Åmic AB), and WO 0146465 (Andersson et al., Gyros AB), which areincorporated by reference herein. In order to secure that the valveand/or anti-wicking means will be located to a desired position and/orhave a desired geometry, appropriate masking is advantageous for anadditional step.

Another variant of step (ii) is to introduce a coat that is non-wettable(hydrophobic coat) on selected parts of the microstructures. Areas onwhich other surface characteristics are desired are then typicallymasked. The non-wettable coat may be introduced for creating localsurface breaks of the same type as indicated in the preceding paragraph.The remaining parts may be intended for liquid transport and thereforetypically need to be processed to surfaces that are wettable byinserting steps according to either alternative (a) or alternative (b)above after step (ii). Remasking for these additional steps is oftenadvantageous for similar reasons as for the first variant. In the casethe uncoated area after unmasking inherently comprises a desiredwettability (either by being wettable or non-wettable), there is no needto introduce any additional surface treatment steps before step (iv).

A third variant of step (ii) is to introduce a coat that is sufficientlywettable or sufficiently non-wettable, but not with sufficiently lownon-specific adsorption (anti-fouling), or vice versa. In this case, anadditional step according to alternative (a) or (b) may be used tomodify the coat to exhibit the missing characteristics while at the sametime retaining an essential part of the surface characteristics createdin step (ii). In this case the same masking can be utilized for the twocoating steps. Demasking and remasking between step (ii) and anadditional step may then not be required.

B. The Substrates

Each of the two planar substrates may comprise microstructures in theform of projections and/grooves as discussed above. In the preferredvariants, however, only one of the two substrates comprisesmicrostructures that then are in the form of open microchannelstructures or open sections of the microchannel structures. The othersubstrate is used to cover these open structures. Either one or both ofthe substrates may have through-going holes that are associated withindividual microchannel structures. These holes may be used as inlets oroutlets for liquids and/or as inlet or outlet vents for air. In the casedifferent sections of a microchannel structure are defined betweendifferent pairs of substrates this kind of holes may providecommunication between the different sections.

The substrates may be made from inorganic or organic material. Typicalinorganic materials are silicon, quartz, glass, etc. Typical organicmaterials are polymer materials, for instance plastics includingelastomers, such as silicone rubber (for instance poly dimethylsiloxane) etc. Polymer material as well as plastics comprises polymersobtained by condensation polymerization, polymerization of unsaturatedorganic compounds and/or other polymerization reactions. Themicrostructures may be created by various techniques such as etching,laser ablation, lithography, replication by embossing, moulding,casting, etc. Each substrate material typically has its preferredtechniques.

From the manufacturing point of view, substrates exposing surfaces andmicrostructures in plastics are many times preferred because the costsfor plastics are normally low and mass production can easily be done,for instance by replication. Typical manufacturing processes involvingreplication are embossing, moulding, casting, etc. See for instance WO9116966 (Pharmacia Biotech AB, Öhman & Ekström), which is incorporatedherein by reference. At the priority date of this invention, thepreferred plastics were polycarbonates and polyolefins based onpolymerizable monomeric olefins that comprise straight, branched and/orcyclic non-aromatic structures. Typical examples are ZeonexTM andZeonorTM from Nippon Zeon, Japan. This does not outrule the use of otherplastics, for instance based on styrenes, methacrylates and/or the like.Suitable polymers may be copolymers comprising different monomers, forinstance with at least one of the monomers discussed above.

C. Plasma Variables and the Gas Plasma Reactor

The electric excitation field applied typically has a frequency in theradiowave or microwave region, i.e., kHz-MHz or GHz respectively. Themodification on the polymer surface caused by the plasma will dependmainly on a number of internal plasma parameters such as: type ofspecies present in the plasma, spatial distributions, energydistributions and directional distributions. The species typicallyderives from one or more organic precursor compounds. In turn theseparameters depend in a complex way on the external plasma parameters:reactor geometry, type of excitation, applied power, type of processgas, gas pressure and gas flow rate.

The results of a treatment may depend on the design of the reactorvessel used meaning that the optimal interval to a certain degree mayvary from one reactor design to another. The results may also depend onwhere in the reactor the surface is placed during the treatment.

A suitable reactor vessel should enable electric excitation power inputfor instance in the microwave or radio wave ranges. The requiredintensity of the plasma may depend on the variables discussed above.Satisfactory gas plasmas may be found in the case the electricexcitation power applied is ≦300 W, with preference for ≦100 W. Thepressures are typically ≦200 mTorr, with preference for ≦100 mTorr. Thedesign of the reactor vessel enables introduction of the vapor phase ofthe organic precursor into the reactor chamber. This includes the optionof heating of the reactor chamber and/or flask containing the organicprecursor. The reactor vessel is designed to facilitate homogenousplasma distribution in the reactor chamber. More details on parametersinfluencing plasma polymerization can be found in Inagaki, N., “Plasmasurface modification and plasma polymerization.” Technomic Publishingcompany, Inc., USA, 1996.

The proper combination of different plasma and apparatus parameters istypically found by varying the values for one or more of theseparameters and study how this affect the properties of the modifiedsubstrate surface, i.e., the resulting hydrophilicity, hydrophobicity,anti-fouling, stability, etc.

D. The Chemical Structure of the Coat

The chemical structure of the coat such as degree and type ofcross-linking, swelling, kinds of functional groups exposed to asurrounding liquid, etc. determines the chemical surfacecharacteristics, primarily wetting/non-wetting ability includinghydrophilicity and hydrophobicity, and non-specific adsorption ofvarious compounds such as proteins and/or other biopolymers andbioorganic compounds.

Surface characterisation of the coat can be carried out by a number ofmethods, such as X-ray photoelectron microscopy (XPS), static secondaryion mass spectrometry (static SIMS), liquid contact angle methods,atomic force microscopy (AFM), near edge X-ray adsorption fine structure(NEXAFS), FTIR and chemical derivatization. For a review see Johnston etal. (J. Electron Spectroscopy and Related Phenomena 81 (1996) 303–317).

Preferably, a sufficiently hydrophilic coat exposes neutral hydrophilicgroups to a liquid in contact with the coat, in particular lower alkylether, such as ethylene oxy, hydroxy groups, etc., and is essentiallyfree of aromatic structures. The coat is essentially free of charged orchargeable groups, in particular if a low non-specific adsorption isrequired. Chargeable groups are karboxy (—COOH), amino (—NH₂), etc.).Non-chargeable groups are hydroxy bound to sp³-hybridized carbon, ether,amido, etc.

There is a relatively large number of publications related to chemicalstructure of polymeric films deposited from gas plasmas that are basedon organic precursor compounds (e.g., U.S. Pat. No. 5,153,072 (Ratner etal.) and U.S. Pat. No. 5,002,794 (Ratner et al.). A general idea hasbeen that the incorporation of groups and/or properties that derive froma precursor compound can be related to the rate of fragmentation in theplasma and the rate of deposition of the coat on a substrate surface. Ithas been discussed that a lower power may decrease fragmentation andincrease the incorporation of groups and properties that derive from theprecursor compound. It has also been discussed that fragmentation of theprecursor compound depends on W/FM where W is the RF power applied, andF and M are the flow rate and the molecular weight, respectively, of theorganic precursor compound. Other variables that have been studied are:(a) the effect of pulsed radiofrequency (RF) discharges on fragmentationof the precursor compound in relation to an increase of the presence ofprecursor structures in the deposited coat, (b) the location of thesubstrate in the gas plasma reactor with the idea that a locationadjacent but not submersed in the plasma will increase the degree ofprecursor structures in the coat, etc. An increase in precursorstructures in a deposited coat has also been suggested if there is anegative temperature gradient between the plasma and the substrate to besurface modified. See Ohkubo et al. (J. Appl. Polym. Sci 41 (1990)349-), López et al. (Langmuir 7 (1991) 766-, D'Agostino et al. (J.Polym. Sci. Part A: Polym. Chem. Edn. 28 (1990) 3378-, Cho et al. (J.Appl. Polym. Sci. 41 (1990) 1373-, Ward et al. (Short, SurfasceInterface Anal. 22 (1994) 477-, Kiaei et al. (J. Biomater. Sci.: Polym.Edn. 4 (1992) 35-, and Panchalingam et al. (ASAJO J. (1993) M305).

The organic precursor compound typically is polymerizable by which ismeant that the compound is capable of forming a high molecular weightinsoluble aggregate on the surface of the substrate. This may involvetraditional polymerization reactions or take place by degradation,rearrangement and extensive reactions of the precursor compound and/orof the intermediary species formed in the gas plasma.

In order for an organic precursor, compound to function in the presentinvention it must have a sufficiently high vapor pressure at theselected temperature within the plasma reactor. This also means thatprecursor compounds that have a low tendency for hydrogen bonding mayhave advantages compared to precursor compounds of the same size thathave a strong tendency for hydrogen bonding.

Small precursor compounds may also have advantages, e.g., with molecularweights ≦2000 dalton, such as ≦1000 dalton or ≦500 dalton. The advantageof small compounds and compounds with weak or no tendency for hydrogenbonding is based on the fact that hydrogen-bonding and increasedmolecular weight tends to increase the boiling point and the vaporpressure.

For hydrophilic coats, suitable precursor compound can be found amongstorganic compounds that have a high content of heteroatoms selectedamongst oxygen, nitrogen and sulphur, provided that the other plasmaparameters are properly set. By the term “high content” in this contextis meant that the ratio between the total number of the heteroatoms,e.g. oxygen, and the number of carbon atoms should be ≧0.1, such as≧0.25 or ≧0.5 or ≧0.75, in the precursor compound. From theoreticalconsiderations, this ratio is never larger than 2. In the case that theorganic precursor compound has certain properties that one would like toincorporate into a coat, but a low content of heteroatoms, this may becompensated for by including oxygen in the gas plasma. Alternatively,one may include one or more other organic compounds for which thecontent of heteroatoms is higher than in the desired precursor compound.Typically, compounds for creating hydrophobic coats are hydrocarbons andfluorinated hydrocarbons (e.g., perfluoinated hydrocarbons (PFH))

For hydrophobic coats, suitable precursor compounds can be found amongstorganic compounds having a low content of heteroatoms selected amongstoxygen, nitrogen and sulphur, provided that the other plasma variablesare properly set. A “low content” in this context means that the ratiobetween the number of heteroatoms, e.g., oxygen, and the number ofcarbon atom should be ≦0.75, such as ≦0.50 or ≦0.25 or ≦0.10. In thecase organic precursor compound has certain properties that one wouldlike to incorporate into a coat, but a high content of heteroatoms, thismight be compensated for by including one or more organic compounds forwhich the content of heteroatoms is lower than in the desired precursorcompound.

Suitable precursor compounds may also be found amongst organic compoundsthat contain one, two or more structural units that are present inpolymers that are known to give coats that are resistant to non-specificadsorption. These kinds of precursor compounds are in the innovativemethod combined with gas plasma conditions enabling this property to beretained in the coat deposited on the substrate.

There are a large number of polymers that are known to reducenon-specific adsorption. Typically, they are non-ionic and hydrophilic,i.e., contains a plurality of neutral hydrophilic groups, such ashydroxy, amido, and lower alkoxy including alkyleneoxy (C₁₋₃ inparticular C₂) and alkyl ether groups. See for instance U.S. Pat. No.6,337,212 (Caliper), WO 0147637 (Gyros AB), U.S. Pat. No. 4,680,201(Hjertén), U.S. Pat. No. 5,840,388 (Karger et al.), U.S. Pat. No.5,240,994 (Brink et al.), and U.S. Pat No. 5,250,613 (Bergström et al.),which are incorporated herein by reference. Precursor compounds to beused in this variant of the invention can, thus, be found amongst lowmolecular weight compounds that comprise one or more of these structuralunits that are present in polymers that reduce non-specific adsorption.At the priority date, one of the most promising precursor compoundscomprise the structural unit —(CH₂)_(n)O—, where (a) n is an integer1–3, with preference for 2, (b) the free valence at the carbon binds tohydrogen or an oxygen, and (c) the free valence at the oxygen binds to ahydrogen or a carbon. The carbon may be sp³⁻, sp²⁻ or sp-hydridized andmay thus be part of a saturated or unstarurated hydrocarbon group suchas alkyl (for instance C₁, C₂, C₃ to C₅) and alkenyl, such as vinyl).This is in-line with the findings of Ratner et al. (U.S. Pat. No.5,002,794 and U.S. Pat. No. 5,153,072) and Timmons et al. (U.S. Pat. No.6,329,024, U.S. Pat. No. 5,876,753, and EP 896035), which areincorporated by reference for precursor compounds comprising 1–4repetitive ethylene oxide units either in straight form or in cyclicform (crown ethers). According to the same principles, one can envisagethat other suitable candidate precursor compounds can be found amongstlow molecular weight compounds which comprise structural units selectedamongst —CH₂OH, —CH₂ (OCH₃), and [—CH₂—CH (OH)]_(n)′—, and [—CH₂—CH(OR)]_(n)′—and corresponding monomers wherever applicable, where (a) n′ isan integer 1–10 with preference for 1–5, (b) R is lower alkyl (C₁₋₅),such as methyl, or lower acyl (C₁₋₅, such as formyl or acetyl), and (c)the free valences binds to atoms selected amongst hydrogen, carbon,sulphur, nitrogen and oxygen. None of sulphur, nitrogen and oxygen bindsa hydrogen when two or more of them binds to the same carbon. Othercandidate precursor compounds are monomers or oligomers (2–10, such as2–5, repeating monomeric units) corresponding to polymers that givecoats that have low non-specific adsorption.

In preferred variants, a coat providing low non-specific adsorption canalso have a sufficient hydrophilicity in order to secure a reliable andreproducible transport of reagents by an aqueous liquid flow. One can,thus, envisage that candidates of precursor compounds can also be foundamong the precursor compounds that are candidates for the creation ofhydrophilic coats. See above.

The thickness of the coat can be <50%, for instance ≦20% or ≦10%, of thesmallest distance between two opposing sides of a microchannel partcomprising the innovative coat. An optimal thickness is typically be≦1000 nm, for instance ≦100 nm or ≦50 nm, with the provision that thecoat shall permit a desired flow to pass through. A lower limit istypically 0.1 nm. The figures of present invention refer to thicknessafter saturation with the liquid intended to pass through a microchannelpart comprising the coat. The coat may or may not swell in contact withthe liquid, which is passing through a microchannel structure.

It is important to control the selected process parameters so that theylead to predetermined surface characteristics, for instance preselectedwetting/or non-wetting properties and/or ability to reduce non-specificadsorption (anti-fouling). This can be accomplished as outlined in theexperimental part that describes the determination of a) liquid contactangles, and b) adsorption of albumin, which is a measure of non-specificadsorption. Once the proper values/ranges of the process parameters havebeen found for the predetermined surface characteristics, the processcan be run without testing.

For aqueous solutions the term “a reduction in non-specific adsorption”(anti-fouling effect) refers to bovine serum albumin as areference/model substance and means that the ratio between adsorption ofbovine serum albumin after and before a gas plasma treatment of asurface according to the invention is ≦0.75, such as ≦0.50 or ≦0.25(decrease ratio). The ratio can be even lower, for instance ≦0.10.

E. Adhering the Substrate Surfaces

There are a number of techniques suggested in the literature. Thusconventional bonding without use of a particular adhesive may beutilized, for example, in the case that the substrates are made ofinorganic material such as silicon, glass, quartz and the like. In thecase that the substrate surfaces comprise plastics, the two surfaces canbe fixed to each other by pressing the surfaces together while heatingselectively the surface not containing microstructures above itstransition temperature, while the surface with the microstructures aremaintained below its transition temperature. In other alternatives,various kinds of adhesives or glues may be used. See further WO 9424900(Ove Öhman), WO 9845693 (Soane et al.), U.S. Pat. No. 6,176,962 (Soaneet al.), WO 9956954 (Quine), and WO 0154810 (Derand et al., Gyros AB),which are incorporated herein by reference. Thermolaminating isimportant because this technology has been shown to be capableminimizing destruction of differences in chemical surfacecharacteristics that are to be retained in the microfluidic deviceobtained after step (iv). See WO 0154810 (Derand et al., Gyros AB).

Problems with so-called bond voids can be minimized if the openmicrochannel structures in a substrate surface is defined by wallsarising from the surface. See WO 9832535 (Lindberg et al.) and WO0197974 (Chazan et al., Caliper).

In order to avoid that an adhesive is pressed into a microchannel duringsteps (iv) and (v) the microchannel structures are preferably defined byrelief patterns that are present in either one or both of the substratesurfaces as outlined in WO 03055790 (Dérand et al.), which isincorporated by reference.

In principle the adhesive may be selected as outlined in U.S. Pat. No.6,176,962 and WO 9845693 (Soane et al.), which are incorporated byreference. Thus, suitable bonding materials include elastomeric adhesivematerials and curable bonding materials. These kinds of bonding materialas well as others may be in liquid form when applied to a substratesurface. Bonding materials including adhesives thus comprises liquidcurable adhesive material and liquid elastomeric material. Afterapplication, the adhesive material is rendered more viscous ornon-flowable for instance by solvent removal or partial curing beforethe other substrate is contacted with the adhesive. The term “liquidform” includes material of low viscosity and material of high viscosity.Curable adhesive includes polymerizable adhesives and activatableadhesives. Thermo-curarable, moisture-curable, and bi-, three- andmulti-component adhesives are also examples of curable adhesives.

III. The Microfluidic Device

This aspect of the invention is primarily characterized in that a partof the inner surface of at least one of the microchannel structures hasbeen modified by the use of gas plasma comprising an organic precursorcompound selected according to the principles outlined for the firstaspect, i.e. has one or more surface characteristics that is achievableby a plasma polymerization coating method. Additional characteristicfeatures are defined below.

The microfluidic device preferably contains a plurality of microchannelstructures, each of which is defined between two or more planarsubstrates. Each microchannel structure may comprise one, two, three ormore functional parts selected among: a) applicationchamber/cavity/area, b) conduit for liquid transport, c) reactionchamber/cavity; d) volume defining unit; e) mixing chamber/cavity; f)chamber for separating components in the sample, for instance bycapillary electrophoresis, chromatography and the like; g) detectionchamber/cavity; h) waste conduit/chamber/cavity; i) internal valve; j)valve to ambient atmosphere; etc. Many of these parts may have one ormore functionalities. There may also be collecting chambers/cavities inwhich a compound, which has been separated, formed or otherwiseprocessed in a microchannel structure are collected and transferred tosome other instrument, for instance an analytical instrument such as amass spectrometer. In addition, there are also one or more outlet ventsfor air. Inlets and outlets for liquids may also function as vents(inlet vent or outlet vent).

The preferred devices are typically disc-shaped with sizes/surface areasand/or forms similar to the conventional CD-format, e.g., their surfaceareas are in the interval from 10% up to 300% of the surface area of aCD of the conventional CD-radii. The upper and/or lower sides of thedisc may or may not be planar.

The preferred microfluidic discs have an axis of symmetry (Cn) that isperpendicular to the disc plane, where n is an integer ≧2, 3, 4 or 5,preferably ∞ (C∞). In other words the disc may be rectangular, such assquare-shaped, or have other polygonal forms, but is preferablycircular. Once the proper disc format has been selected centrifugalforce may be used for driving liquid flow. Spinning the device around aspin axis that typically is perpendicular or parallel to the disc planemay create the necessary centrifugal force. In the most obvious variantsat the priority date, the spin axis coincides with the above-mentionedaxis of symmetry.

Different principles may be utilized for transporting the liquidaliquots within the microfluidic device/microchannel structures betweentwo or more of the functional parts described above. Inertia force maybe used, for instance by spinning the disc as discussed in the precedingparagraphs. Other forces that may be used are electrokinetic forces andnon-electrokinetic forces, such as capillary forces, hydrostaticpressure, etc. In preferred variants utilizing centrifugal force forliquid transport, each microchannel structure comprises an upstreamsection that is at a shorter radial distance than a downstream sectionrelative to a spin axis.

The microfluidic device may also comprise common channels connectingdifferent microchannel structures, for instance common distributionchannels for introduction of liquids and common waste channels includingwaste reservoirs. Common channels including their various parts such asinlet ports, outlet ports, vents, etc., are considered to be part ofeach of the microchannel structures they are connecting. Commonmicrochannels may also fluidly connect groups of microchannel structuresthat are in different planes or in the same plane.

IV. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Plasma Treatment with Diethylene Glycol Dimethyl Ether

A polycarbonate CD disc (Macrolon DP-1265, Bayer AG, Germany), andpieces cut from a polycarbonate CD disc were placed in a plasma reactor(CVD Piccolo, Plasma Electronic, Germany), and subjected to argon plasmatreatment at 24 W for 2 min. Subsequently, the polycarbonate surfaceswere treated with plasma of diethylene glycol dimethyl ether (diglyme;Aldrich, USA) at 24 W for 5 min. The water contact angle (sessile drop)of the resulting surfaces was measured on a Ramé-Hart manual goniometerbench. The average of six equilibrium measurements (three droplets) was48°.

Example 2 Plasma Treatment with Diethylene Glycol Dimethyl Ether andAllylic Alchohol

A polycarbonate CD disc (Macrolon DP-1265, Bayer AG, Germany), andpieces cut from a polycarbonate CD disc were placed in a plasma reactor(CVD Piccolo, Plasma Electronic, Germany), and subjected to argon plasmatreatment at 24 w for 2 min. Subsequently, the polycarbonate surfaceswere treated with plasma of diethylene glycol dimethyl ether (diglyme;Aldrich, USA) at 24 W for 5 min. Finally, they were subjected to plasmaof allylic alcohol (Merck, Germany) at 12 W for 5 min. The water contactangle (sessile drop) of the resulting surfaces was measured on aRamé-Hart manual goniometer bench. The average of six equilibriummeasurements (three droplets) was <10°.

Example 3 Plasma Treatment with Ethylene Glycol Vinyl Ether

A polycarbonate CD disc (Macrolon DP-1265, Bayer AG, Germany), andpieces cut from a polycarbonate CD disc were placed in a plasma reactor(CVD Piccolo, Plasma Electronic, Germany), and subjected to argon plasmatreatment at 24 w for 2 min. Subsequently, the polycarbonate surfaceswere treated with plasma of ethylene glycol vinyl ether (Aldrich, USA)at 12 W for 5 min.

The water contact angle (sessile drop) of the resulting surfaces wasmeasured on a Ramé-Hart manual goniometer bench. The average of sixequilibrium measurements (three droplets) was 22°.

Example 4 Microfluidic Test

A silicone rubber lid (polydimethylsiloxane) was placed on apolycarbonate CD with recessed microchannel pattern, (50–200 μm wide,50–100 μm deep), that had been treated either with diglyme plasma, orwith diglyme plasma with subsequent allylic alcohol plasma treatment, asdescribed above. Alternatively, silicone rubber with recessedmicrochannel pattern (1000 μm wide, 100 μm deep) was placed on flatpolycarbonate surfaces that had been treated either with diglyme plasma,or with diglyme plasma with subsequent allylic alcohol plasma treatment,as described above. Resulting flow channels were examined using asolution of Cibacron Brilliant Red (CIBA limited) in MilliQ water(Millipore). A drop was placed at the channel inlet and it was concludedthat flow rate into channels on surfaces that had been subjected todiglyme plasma with subsequent allylic alcohol plasma treatment wassignificantly higher than on surfaces that had only been treated withdiglyme plasma.

Example 5 Protein Adsorption Studied with Total Internal ReflectionFluorescence (TIRF) Spectroscopy

The theory of TIRF spectroscopy, as well as the experimental set-up usedin the present work is described in Example 1.

Bovine serum albumin (BSA; fraction V, Sigma, USA) was chosen as modelprotein for adsorption studies, and labelled withfluorescein-5-isothiocyanate (FITC; isomer I; Molecular Probes), asdescribed in [Lassen, B. and Malmsten, M., Competitive proteinadsorption studied with TIRF and ellipsometry. Journal of colloid andinterface science, 1996. 179: p. 470–477]. The molar ratio of FITC toproteins was found to be approximately unity in all cases.

A TIRF fluorescence intensity graph resulting from adsorption of 400 ppmFITC-BSA on untreated polycarbonate (PC) is shown in FIG. 1, togetherwith a graph representing the same experiment on a diglymeplasma-treated surface. TIRF fluorescence intensity graphs using diglymeplasma+allylic alcohol plasma (FIG. 2), and ethylene glycol vinyl etherplasma (FIG. 3) are also presented here.

It is apparent from the figures that the ratio between adsorption ofprotein on the treated surface and the untreated surface always is<0.25.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

1. A method for the manufacture of a microfluidic device comprising oneor more enclosed microchannel structures, each of which comprises asection that is defined between two essentially planar substrates,wherein one surface in either one or both of the substrates comprisesmicrostructures in the form of grooves or projections that match eachother so that they together define said section for each of said one ormore microchannel structures when the two surfaces are apposed in themicrofluidic device, the method comprises the steps of: (i) providingthe planar substrates, (ii) placing at least one substrate in a gasplasma reactor, and creating within said plasma reactor a gas plasmacontaining an organic precursor compound, said organic precursorcompound and the conditions in the reactor are selected such that awettable coat that is also anti-fouling is formed on a selected part ofthe surface of the substrate, iii) removing the substrate from theplasma reactor, and (iv) adhering the surfaces of the substrates to eachother so that said section of each of said microchannel structures isformed between the two surfaces.
 2. The method of claim 1, wherein saidsection is a complete microchannel structure.
 3. The method of claim 1,wherein the precursor compound and the conditions in the reactor areselected to give the wettable coat in step (ii) a water contact angle≦90°.
 4. The method of claim 3, wherein the water contact angle is ≦60°.5. The method of claim 1, wherein the precursor compound and theconditions are selected in step (ii) so that the wettable coat isintroduced on selected parts of individual microchannel structures, andthat a second coat is introduced on other selected parts of themicrochannel structures by an additional coating step introduced betweensteps (ii) and (iv).
 6. The method of claim 5, wherein the second coatis non-wettable.
 7. The method of claim 5, wherein the second coat iswettable.
 8. The method of claim 1, wherein the coat introduced in step(ii) has been modified by an additional step between step (ii) and step(iii) also utilizing a gas plasma.
 9. The method of claim 8, wherein theadditional step strengthens the wettability of the coat withoutessentially destroying the anti-fouling property accomplished in step(ii).